The overall objective of this proposal is to define in vitro methods for inducing MHC alloantigen-specific nonresponsiveness in resting unprimed T cells and to assess the extent to which these cells remain capable of causing GVHD. This objective follows from preliminary data demonstrating that the secondary MLC response of human T cells can be ablated when the primary MLC is carried out in the presence of a CD3-specific monoclonal antibody. These results have suggested novel strategies for GVHD prevention, particularly in the setting of MHC-mismatched transplantation. Further development and characterization is necessary, however, before these strategies can be tested in clinical trials. Different methods for achieving specific alloantigen-nonresponsiveness must first be optimized and compared, and more importantly, these methods should be tested in an animal model in order to assess the potential success and limitations of these approaches for GVHD prevention. The first specific aim of this project is to develop and evaluate in vitro methods for inducing MHC alloantigen-specific nonresponsiveness in resting unprimed human T cells. This aim will be approached by two distinct methods: a) MLC in the presence of CD3 antibody and b) depletion of the alloactivated subset in MLC. Experiments will be carried out to measure the frequency of residual MHC class II-alloresponsive cells and to assess whether MHC-class I-restricted cytotoxic precursors are made nonresponsive by these methods. The second specific aim is to develop and evaluate in vitro conditions for inducing MHC alloantigen-specific nonresponsiveness in resting unprimed murine T cells. This will be accomplished by developing methods parallel to those found to be successful for human cells. The third specific aims is to evaluate whether murine T cells made nonresponsive to allogeneic cells by in vitro methods remain capable of causing GVHD in recipients with disparity for a) MHC class I or class II antigens, b) minor histocompatibility antigens, or c) for various combinations of MHC and minor antigens. Approaches successful for decreasing the risk of GVHD in the murine model will ultimately be tested in human marrow transplantation. Selective removal or inactivation of T cells reactive against host alloantigens may avoid the problem of graft failure associated with methods that deplete T cells from donor marrow irrespective of their antigen specificity. Furthermore, administration of host-nonresponsive donor T cells may facilitate posttransplant immune reconstitution.